Rapid identification of Ralstonia pickettii using PCR-nucleic acid test strips

Abstract

To develop a method for rapid detection of Ralstonia pickettii in water for pharmaceutical purpose using PCR-nucleic acid test strips. The genomic DNA of Ralstonia pickettii was extracted by boiling method. A pair of specific primers targeting the 16S rDNA with FITC and biotin labeling of the 5' ends was designed and cloned into competent E. coli DH5α cells. The nucleic acid test strips were assembled, and the workload of streptavidin labeled with colloidal gold and antibody concentration in the reaction system was optimized. After verification of the reaction mechanism and assessment of the test sensitivity, specificity and stability, the test strip was used for detecting 7 known strains of Ralstonia pickettii detected in pharmaceutical water, and an evolutionary tree was constructed to analyze the source of contamination. The genomic DNA extracted by boiling method had a purity between 1.8 and 2.0, and the PCR products showed a 100% similarity of with Ralstonia pickettii 16S rDNA registered in GenBank. Using the colloidal gold amplification principle, in every 100 μL colloidal gold solution, 3.5 μL streptavidin was added; the detection line on nitrocellulose membrane was 2.0 mg·mL-1 anti FITC antibody, and the quality control line was 1.2 mg · mL-1 biotinylated BSA, and they generate a red band after binding with positive amplification product. Specificity test of the assembled test strip yielded consistent result with agarose gel electrophoresis without cross reaction with Acinetobacter, Aeromonas, Pseudomonas, or Leclercia adecarboxylata. Sensitivity test of the strip showed a lower detection limit for DNA concentration of 10-5 ng/μL, with a sensitivity 1000 times that of agarose gel electrophoresis. The test strip still had good performance after storage for 3, 6, 9 and 12 months. We successfully developed a PCR-nucleic acid test strip for convenient and cost-effective detection of Ralstonia pickettii with good specificity and sensitivity and low cost to facilitate daily monitoring of pharmaceutical water contaminations.